Cardiolipin (CL) is an essential phospholipid for normal mitochondrial energy metabolism so that physiological and pathological perturbations in its levels result in alterations in the structure, function and assembly of mitochondria. CL is an integral part of most of the components of the mitochondrial energy transducing system. Myocardial dysfunction and apoptosis are associated with abnormal CL levels in Barth Syndrome, aging, ischemia/reperfusion, and heart failure. Saccharomyces cerevisiae mutants (crd1?) lacking CL, but containing its precursor phosphatidylglycerol (PG), or mutants (pgs1?) lacking both PG and CL display similar phenotypes to mammalian cells with reduced PG and CL levels or the inability to make PG and CL, respectively. Therefore, yeast is an excellent model system to study their role of these lipids in mitochondrial function. Studies of yeast pgs1? and crd1? mutants led to two findings that are the basis for the proposed studies. (1) Translation repressors that bind 5'of the translation initiation site on mRNA for subunit 4 (Cox4p) of the mitochondrial electron transport chain component cytochrome c oxidase (Complex IV) are specifically activated or synthesized in response to altered mitochondrial phospholipid composition in pgs1? mutants. Studies are proposed to genetically and biochemically define the components and mechanism of this novel mitochondrial stress response pathway. (2) Complex IV and Complex III (cytochrome bc1) form a III2IV2 supercomplex that kinetically behaves as a single unit "respirasome", which was established by using a crd1? mutant to be specifically dependent on CL. Experiments are proposed to determine the phospholipid stoichiometry in the individual Complexes III and IV and in the III2IV2 supercomplex and the location of CL within the latter required for formation and stabilization of the supercomplex. A 3-D structure of the III2IV2 supercomplex has been determined by negative stain-electron microscopy and single particle analysis, which will be refined using cryoelectron microscopy to establish how Complex III and IV are organized in the supercomplex. Phospholipid analysis and structural data will be integrated to establish how CL is involved in "gluing" together the supercomplex components. Defining the roles PG and CL play in normal mitochondrial function will shed light on the molecular basis for cellular dysfunction in physiological and pathological states where these lipids are reduced.